Vanilloids (also referred herein as “vanilloid compounds”) are defined as chemical compounds derived from a vanillyl group, the latter being formed by a benzyle group substituted with a hydroxyle and a methoxy group, and whose chemical structure is shown here below:
wherein R is generally selected from the group consisting of H, a lower alkyl such as a methyl (—CH3) or an ethyl (—CH2CH3), an aldehyde and a carboxylic acid. Optionally, the —OH group is substituted with an O-glycosyl group.
In a non-limitative manner, vanilloids include vanillyl alcohol, vanillin, vanillic acid, vanillin-glycoside, acetovanillon, vanillylmandelic acid, homovanillic acid, and capsaicidoids such as capsaicin.
Among vanilloids, vanillin of chemical name 4-hydroxy-3-methoxybenzaldehyde is one of the most important aromatic flavor compound used in foods, beverages, fragrance, pharmaceuticals and polymers. Vanillin was historically extracted from Vanilla planifolia, Vanilla tahitiensis and Vanilla pompona pods. The demand getting higher, today, less than 5% of worldwide vanillin production comes from natural vanilla pods. Currently, chemical synthesis is the most important process for producing vanillin.
There is a growing interest in other sources of vanillin and in particular in bioconversion processes. The use of microbial cells and their enzymes as biocatalysts for the synthesis of chemicals and flavor compounds have attracted much attention lately. Advantageously, under certain criteria, the products of such bioconversion may be considered ‘natural’ by legislations, such as the European Community one.
Bioconversion processes are based on the following substrates: lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, sugars, phenolic stilbenes, waste residues and aromatic amino acids. The recent review from Kaur and Chakraborty (Kaur B, Chakraborty D. “Biotechnological and molecular approaches for vanillin production: a review.” Appl Biochem Biotechnol. 2013 February; 169(4):1353-72) lists several biosynthetic pathways and appropriate cells used for bioconversion of vanilloids.
De novo synthesis from glucose using metabolically engineered yeast strains has been recently described (Hansen et al., De Novo Biosynthesis of Vanillin in Fission Yeast (Schizossacharomyces pombe) and Baker's Yeast (Saccharomyces cerevisae); Appl. Environ. Microbiol. 2009, 75(9):2765). The engineered pathway involves a 3-dehydroshikimate dehydratase (3DSD), an aromatic carboxylic acid reductase (ACAR) and an O-methyltransferase (COMT), as shown in FIGS. 1 and 2 (see in particular FIG. 2, and pathways “1” and “2”), which relate to the global reaction scheme of vanillin biosynthesis from glucose. In S. cerevisiae, the ACAR enzyme requiring activation mediated by a phosphopantetheinyl transferase, this enzyme was also introduced.
So far, studies related to recombinant unicellular hosts capable of producing vanilloids had mostly focused on the use of O-methyltransferases of the catechol methyltransferase type (EC 2.1.1.6; CAS no 9012-25-3), which are known to catalyze the methylation of catechol into guaiacol. The catechol O-methyltransferase accepts flavanols like epicatechin and epigallocatechin, catecholamines like L-DOPA and adrenalin, 3,4-dihydroxyphenylacetic acid, caffeic acid as substrates.
Later, the same authors improved their pathway by using mutants of human catechol acid O-methyltransferase having a better specificity, thereby limiting the production of iso-vanillin (WO 2013/022881).
Other improvements of vanillin biosynthesis pathway have been proposed, and in particular:                Alcohol dehydrogenases ADH6 & ADH7 are known to convert vanillin into its corresponding vanillyl alcohol. Studies have suggested the deletion of the adh6 gene in vanillin-producing yeasts (Hansen et al., 2009).        Brochado (Brochado et al., 2010) suggested the deletion of genes encoding pyruvate decarboxylase (PDC1) and glutamate dehydrogenase (GDH1), since the deletion of these genes increases the availability of co-factors (ATP, NADPH . . . ) for the biosynthesis pathway of vanilloids.        Most of phenolic compounds such as vanillin show some toxicity for many living organisms with increased concentration of vanilloids. In case of Saccharomyces cerevisiae, growth defect is significant with concentrations as low as 0.5 g/l. To avoid the impaired growth of producing microorganisms, it has also been proposed to isolate the produced vanilloids from the culture medium, in particular with resins. Another suggested solution is promoting conversion of vanillin into vanillin β-D-glucoside, this glycosylation inhibiting its toxic effect (Hansen et al., 2009; Brochado et al., 2010).        
For comprehension purposes, FIGS. 2A and 2B summarizes the three main vanillin biosynthesis pathways. According to the invention, pathway “1” and pathway “2” are part of the “dehydroshikimic acid pathway”. Within said dehydroshikimic acid pathway, pathway “1” represents a first alternative route and will be referred herein as the “AAD-dependent dehydroshikimic acid pathway”. Pathway “2” represents a second alternative route, and will be referred herein as the “ACAR-dependent dehydroshikimic acid pathway”.
The term “3-dehydroshikimic acid” also called 3-DHS designates the compound of the systemic name (4S,5R)-4,5-D-dihydroxy-3-oxocyclohexene-1-carboxylic acid.
Another pathway for producing a vanilloid in yeast is inspired from the natural biosynthesis pathway observed in the Vanilla planifolia orchid, such as the one described in patent application US 2003/0070188. This pathway, shown in FIG. 2 (pathway “3”), uses aromatic amino acids such as phenylalanine and tyrosine as primary substrates.
The term “aromatic amino acids” designates amino acids that include an aromatic ring. Among the twenty standard amino acids, four of them are aromatic: phenylalanine, tryptophan, histidine and tyrosine.
However, even if these pathways are effective for producing vanilloids in recombinant hosts such as yeasts, they do not allow a sufficient production of these compounds for being industrialized. Different propositions have been made to improve the production of vanilloids by fermentation in cells.
The production of vanillin in metabolically engineered yeast strains is hindered by the production of other vanilloids which may be undesirable, or to the least less desirable because of, for instance, a lack of pronounced aromatic flavor. In particular, the production of vanillin in S. pombe leads to the production of different sorts of vanilloid compounds such as vanillin, isovanillin, vanillyl alcohol, isovanillic acids and/or vanillic acids (see Hansen et al., 2009). Indeed isovanillin and isovanillic acids are closely related compounds which may be produced in large amounts during the production of Vanillin involving O-methyltransferases. Such production in the recombinant host is problematic, because isovanillin does not share the same aromatic properties as its counterpart.
Unfortunately, the ratio vanillin/isovanillin which is obtained using currently available engineered yeast strains is equal or inferior to 125:1, which means that those pathways are not 100% selective for vanillin production.
Thus, there remains a need for novel methods for producing vanilloid compounds in a recombinant unicellular host.
There also remains a need for improving the production of vanilloid compounds, and decreasing the production of isovanillin.
There also remains a need for improving selectivity of this production in a recombinant unicellular host such as yeast towards vanilloid compounds of interest, such as vanillin.
Thus there also remains a need for producing a substantially pure vanilloid compound with a recombinant unicellular host.